RNAi modulation of SCAP and therapeutic uses thereof

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a SCAP gene (Human SCAP gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of a SCAP gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by Human SCAP expression and the expression of a SCAP gene using the pharmaceutical composition; and methods for inhibiting the expression of a SCAP gene in a cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.13/080,334 filed on Apr. 5, 2011, which is a continuation of Ser. No.12/749,159 filed on Mar. 29, 2010 and issued as U.S. Pat. No. 7,919,613on Apr. 5, 2011, which is a divisional of U.S. patent application Ser.No. 11/857,120 filed on Sep. 18, 2007 and issued as U.S. Pat. No.7,737,266 on Jun. 15, 2010, which claims priority to U.S. ProvisionalApplication No. 60/845,289, filed Sep. 18, 2006. The entire contents ofthese applications are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns methods of treatment using modulators ofthe gene SREPB cleavage activating protein (SCAP). More specifically,the invention concerns methods of treating disorders associated withundesired SCAP activity, by administering short interfering RNA thatdown-regulate the expression of SCAP, and agents useful therein.

BACKGROUND OF THE INVENTION

Lipid homeostasis is essential to all living beings that rely on lipidmembranes to separate their cell's vital functions from the environment,including all animals, and humans. Furthermore, lipids are used asenergy reservoirs by many organisms. A vast array of different lipidicsubstances, including, for example, phospholipids, triglycerides, fattyacids, and sterols, perform a wide variety of essential functions incells. Altogether, lipid homeostasis is a tightly regulated,multi-branched, intricate web of interdependent processes in essentiallyall higher organisms.

Naturally, the more complex a system, the more can go awry. A largenumber of diseases and conditions, e.g. in humans, are known to be, inwhole or in part, consequences of lipid homeostasis dysfunctions. Theseinclude both inherited diseases, where one or a number of the many genesinvolved in lipid homeostasis completely or partially loses itsfunction, or is mis-regulated, as well as acquired diseases, where genefunction or gene regulation in the body is altered after single orrepeated contact with one or a combination of substances.

In many a species including humans, the body's needs for lipids arefilled partially by dietary intake as well as by the synthesis of lipidsfrom precursors. The liver stands out as the single organ responsiblefor the collection of dietary lipid intake, lipid synthesis, and thecontrol of lipid release to and re-uptake from the bloodstream.Consequentially, it is involved in many, if not all, lipid metabolismdisorders.

Many such disorders are caused by, or accompanied with, an overabundanceof certain lipids in all or parts of the body, be it from excessiveintake, faulty degradation or transport, or excessive de novo synthesis.

For example, Non-Alcoholic Fatty Liver Disease (NAFLD) is a conditionwhere excess triglycerides accumulate in the liver, and is associatedwith various drugs, nutritional factors, multiple genetic defects inenergy metabolism, and, most prominently, insulin resistance (Browning JD and Horton J D, J. Clin. Investigation 2004, 114:147). Conversely, ahallmark of atherosclerosis is the appearance of so-called foam cells,macrophages filled with excess cholesterol and cholesterol esters (KnuthH S, Front Biosci 2001, 6:D429). Other non-limiting examples ofdisorders associated with excessive levels of lipids in the body are:non-alcoholic liver disease, fatty liver, hyperlipemia, hyperlipidemia,hyperlipoproteinemia, hypercholesterolemia and/or hypertriglyceridemia,atherosclerosis, pancreatitis, non-insulin dependent diabetes mellitus(NIDDM), coronary heart disease, obesity, metabolic syndrome, peripheralarterial disease, and cerebrovascular disease. The treatment ofdisorders of this type could potentially be aided by attenuating thebody's own synthesis of lipids.

A central element in the regulation of lipid biosynthesis in the humanliver is a group of transcription factors termed Sterol RegulatoryElement Binding Proteins (SREBPs). There are three SREBP isoforms calledSREBP-1a, SREBP-1c and SREBP-2. They are located in the endoplasmaticreticulum (ER) in a precursor form (Yokoyama C. et al., Cell 1993,75:187; Hua X. et al., Proc. Natl. Acad. Sci. 1993, 90:11603) which, inthe presence of cholesterol, is bound to cholesterol and two otherproteins: SCAP (SREBP-cleavage activating protein) and Insig1(Insulin-induced gene 1). When cholesterol levels fall, Insig-1dissociates from the SREBP-SCAP complex, allowing the complex to migrateto the Golgi apparatus, where SREBP is cleaved by S1P and S2P (site 1/2protease; Sakai J et al, Mol. Cell. 1998, 2:505; Rawson R. B. et al,Mol. Cell. 1997, 1:47), two enzymes that are activated by SCAP. Thecleaved SREBP then migrates to the nucleus and acts as a transcriptionfactor by binding to the SRE (sterol regulatory element) of a number ofgenes and stimulating their transcription (Briggs M. R. et al., J. Biol.Chem. 1993, 268:14490). Among the genes transcribed are theLDL-Receptor, up-regulation of which leads to increased in-flux ofcholesterol from the bloodstream, HMG-CoA reductase, the rate limitingenzyme in de-novo cholesterol synthesis (Anderson et al, Trends CellBiol 2003, 13:534), as well as a number of genes involved in fatty acidsynthesis.

In an attempt to lower the body's own production of lipids, oneattractive option therefore would seem to be the blocking of SREBPactivation. Since SCAP-binding is a prerequisite for the transport andactivation of all three SREBP isoforms, an inhibition of SCAP's activitycould lead to a general down-regulation of cellular lipid synthesis anduptake. For example, SCAP activity could be inhibited by agents bindingto the sterol sensing domain (SSD) of SCAP with higher affinity thancholesterol, and preferably in an irreversible manner, therebyprohibiting SREBP transport and activation. Alternatively, inhibitingthe translation and/or transcription of the gene encoding SCAP couldlead to lower levels of SCAP present in the ER membrane and availablefor SREBP-binding and activation.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in thetreatment of pathological processes mediated by excessive levels oflipids, there remains a need for an agent that can selectively andefficiently attenuate the body's own lipid biosynthesis, e.g byinhibiting SCAP, and thereby SREBP, activity, using the cell's own RNAimachinery. Such agent shall possess both high biological activity and invivo stability, and shall effectively inhibit expression of a targetSCAP gene, such as human SCAP, for use in treating pathologicalprocesses mediated directly or indirectly by SCAP expression.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of a SCAP genein a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases mediated by the expression of a SCAP gene, such as inconditions and diseases associated with excessive levels of lipidsand/or unwanted lipid biosynthesis. The dsRNA of the invention comprisesan RNA strand (the antisense strand) having a region which is less than30 nucleotides in length, generally 19-24 nucleotides in length, and issubstantially complementary to at least part of an mRNA transcript of aSCAP gene. The SCAP gene is preferably a human SCAP gene, and morepreferably a Homo sapiens SCAP gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of a SCAP gene. ThedsRNA comprises at least two sequences that are complementary to eachother. The dsRNA comprises a sense strand comprising a first sequenceand an antisense strand comprising a second sequence. The antisensestrand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding a SCAP gene, and theregion of complementarity is less than 30 nucleotides in length,generally 19-24 nucleotides in length. The dsRNA, upon contacting with acell expressing the SCAP gene, inhibits the expression of the SCAP geneby at least 20%, or at least 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%,70%, 85%, 90% or 95%, e.g. in primary hamster hepatocytes.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that is selected from the group consistingof the sense strand sequences of the RNAi agents AD-9490-AD-9513 (unevennumbers of the group of SEQ ID NO: 1-48, Table 1), and the secondsequence is selected from the group consisting of the antisense strandsequences of AD-9490-AD-9513 (even numbers of the group of SEQ ID NO:1-48, Table 1). The dsRNA molecules of the invention can be comprised ofnaturally occurring nucleotides or can be comprised of at least onemodified nucleotide, such as a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative. Alternatively, themodified nucleotide may be chosen from the group of: a T-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Generally, such modifiedsequence will be based on a first sequence of said dsRNA selected fromthe group consisting of the sense sequences of AD-9490-AD-9513 (Table 1)and a second sequence selected from the group consisting of theantisense sequences of AD-9490-AD-9513 (Table 1).

TABLE 1  RNAi agents selected for the down-regulation of homo sapiens(NM_012235.2), mus musculus (NM_001001144.1) and Cricetus cricetus(U67060) SCAP, and minimal off-target interactions in humans SEQ DuplexID SEQ ID identifier Sense strand sequence¹ NO:Antisense strand sequence¹ NO: AD-9490 gauuggcauccugguauacTT 1guauaccaggaugccaaucTT 2 AD-9491 agcgccucaucauggcuggTT 3ccagccaugaugaggcgcuTT 4 AD-9492 ggccuucuacaaccaugggTT 5cccaugguuguagaaggccTT 6 AD-9493 gaggugugggacgccauugTT 7caauggcgucccacaccucTT 8 AD-9494 uggauuggcauccugguauTT 9auaccaggaugccaauccaTT 10 AD-9495 gccauugucugcaacuuugTT 11caaaguugcagacaauggcTT 12 AD-9496 ccaucacccuggucuuccaTT 13uggaagaccagggugauggTT 14 AD-9497 uguccuuccgccacuggccTT 15ggccaguggcggaaggacaTT 16 AD-9498 ccuucuacaaccaugggcuTT 17agcccaugguuguagaaggTT 18 AD-9499 gaccgcagcacaggcaucaTT 19ugaugccugugcugcggucTT 20 AD-9500 ggauuggcauccugguauaTT 21uauaccaggaugccaauccTT 22 AD-9501 aucugggaccgcagcacagTT 23cugugcugcggucccagauTT 24 AD-9502 ucugcaucuuagccugcugTT 25cagcaggcuaagaugcagaTT 26 AD-9503 agaucgacauggucaagucTT 27gacuugaccaugucgaucuTT 28 AD-9504 caucacccuggucuuccagTT 29cuggaagaccagggugaugTT 30 AD-9505 caucuuagccugcugcuacTT 31guagcagcaggcuaagaugTT 32 AD-9506 ugcaucuuagccugcugcuTT 33agcagcaggcuaagaugcaTT 34 AD-9507 aagaucgacauggucaaguTT 35acuugaccaugucgaucuuTT 36 AD-9508 aggugugggacgccauugaTT 37ucaauggcgucccacaccuTT 38 AD-9509 cagcgccucaucauggcugTT 39cagccaugaugaggcgcugTT 40 AD-9510 ggaccgcagcacaggcaucTT 41gaugccugugcugcgguccTT 42 AD-9511 cugccauugucugcaacuuTT 43aaguugcagacaauggcagTT 44 AD-9512 cugcaucuuagccugcugcTT 45gcagcaggcuaagaugcagTT 46 AD-9513 ucuuagccugcugcuacccTT 47ggguagcagcaggcuaagaTT 48 ¹Capital letters = desoxyribonucleotides; smallletters = ribonucleotides; underlined: nucleoside thiophosphate

In a preferred embodiment, the dsRNA is chosen from the group ofAD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500,AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491, AD-9506, AD-9508,AD-9502, AD-9504, AD-9507, AD-9493, AD-9501, AD-9497, AD-9509 andAD-9513, and inhibits the expression of a SCAP gene in a cell, e.g. aprimary hamster hepatocyte, by at least 20%.

More preferably, the dsRNA is chosen from the group of AD-9505, AD-9498,AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, AD-9492, AD-9499,AD-9496, AD-9510, AD-9511, AD-9491, AD-9506, AD-9508, AD-9502, AD-9504,AD-9507, AD-9493, AD-9501, AD-9497, and AD-9509, and inhibits theexpression of a SCAP gene in a cell, e.g. a primary hamster hepatocyte,by at least 30%.

Yet more preferably, the dsRNA is chosen from the group of AD-9505,AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, AD-9492,AD-9499, AD-9496, AD-9510, AD-9511, AD-9491, AD-9506, AD-9508, AD-9502,AD-9504, AD-9507, and inhibits the expression of a SCAP gene in a cell,e.g. a primary hamster hepatocyte, by at least 40%.

Yet more preferably, the dsRNA is chosen from the group of AD-9505,AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, AD-9492,AD-9499, AD-9496, AD-9510, AD-9511, AD-9491, AD-9506, AD-9508, AD-9502,and inhibits the expression of a SCAP gene in a cell, e.g. a primaryhamster hepatocyte, by at least 50%.

Yet more preferably, the dsRNA is chosen from the group of AD-9505,AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, AD-9492,AD-9499, AD-9496, AD-9510, AD-9511, and inhibits the expression of aSCAP gene in a cell, e.g. a primary hamster hepatocyte, by at least 60%.

Yet more preferably, the dsRNA is chosen from the group of AD-9505,AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, andinhibits the expression of a SCAP gene in a cell, e.g. a primary hamsterhepatocyte, by at least 70%.

Most preferably, the dsRNA is chosen from the group of AD-9505, AD-9498,AD-9512, and inhibits the expression of a SCAP gene in a cell, e.g. aprimary hamster hepatocyte, by at least 75%.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is generally a mammalian cell,such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of a SCAP gene, e.g. a humanor Homo sapiens SCAP gene, in an organism, generally a human subject,comprising one or more of the dsRNA of the invention and apharmaceutically acceptable carrier or delivery vehicle. Therein, thedsRNA is preferably chosen from the group of AD-9505, AD-9498, AD-9512,AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, AD-9492, AD-9499, AD-9496,AD-9510, AD-9511, AD-9491, AD-9506, AD-9508, AD-9502, AD-9504, AD-9507,AD-9493, AD-9501, AD-9497, AD-9509, and AD-9513, and inhibits theexpression of a SCAP gene in a cell, e.g. a primary hamster hepatocyte,by at least 20%. For further preferred embodiments of the pharmaceuticalcomposition, the dsRNA is chosen from the groups given above.

In another embodiment, the invention provides a method for inhibitingthe expression of a SCAP gene, e.g. a human SCAP gene, and preferably aHomo sapiens SCAP gene, in a cell, comprising the following steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoding a SCAP gene, and wherein        the region of complementarity is less than 30 nucleotides in        length, generally 19-24 nucleotides in length, and wherein the        dsRNA, upon contact with a cell expressing the SCAP gene        inhibits expression of the SCAP gene by at least 20%, or at        least 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 85%, 90%        or 95%, e.g. in primary hamster hepatocytes; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        SCAP gene, thereby inhibiting expression of the SCAP gene in the        cell.

Therein, the dsRNA is preferably chosen from the group of AD-9505,AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500, AD-9492,AD-9499, AD-9496, AD-9510, AD-9511, AD-9491, AD-9506, AD-9508, AD-9502,AD-9504, AD-9507, AD-9493, AD-9501, AD-9497, AD-9509, and AD-9513, andinhibits the expression of a SCAP gene in a cell, e.g. a primary hamsterhepatocyte, by at least 20%. For further preferred embodiments of theabove method, the dsRNA is chosen from the groups given above.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes mediated by SCAPexpression, e.g. disorders of lipid metabolism, lipid homeostasis,and/or lipid distribution, e.g. non-alcoholic liver disease, fattyliver, hyperlipemia, hyperlipidemia, hyperlipoproteinemia,hypercholesterolemia and/or hypertriglyceridemia, atherosclerosis,pancreatitis, non-insulin dependent diabetes mellitus (NIDDM), coronaryheart disease, obesity, metabolic syndrome, peripheral arterial disease,and cerebrovascular disease, comprising administering to a patient inneed of such treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the dsRNAs of theinvention. Therein, the dsRNA is preferably chosen from the group ofAD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494, AD-9500,AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491, AD-9506, AD-9508,AD-9502, AD-9504, AD-9507, AD-9493, AD-9501, AD-9497, AD-9509, andAD-9513, and inhibits the expression of a SCAP gene in a cell, e.g. aprimary hamster hepatocyte, by at least 20%. For further preferredembodiments of the pharmaceutical composition, the dsRNA is chosen fromthe groups given above.

In another embodiment, the invention provides vectors for inhibiting theexpression of a SCAP gene in a cell, comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of a SCAP gene in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of a SCAP genein a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by the expression of a SCAP gene usingdsRNA. dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi).

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of a SCAP gene. Theuse of these dsRNAs enables the targeted degradation of mRNAs of genesthat are implicated in diseases involving faulty regulation of disordersof lipid metabolism, lipid homeostasis, and/or lipid distribution, e.g.non-alcoholic liver disease, fatty liver, or various forms ofdyslipidemia. Using cell-based and animal assays, the present inventorshave demonstrated that very low dosages of these dsRNA can specificallyand efficiently mediate RNAi, resulting in significant inhibition ofexpression of a SCAP gene. Thus, the methods and compositions of theinvention comprising these dsRNAs are useful for treating pathologicalprocesses mediated by SCAP expression, e.g. disorders of lipidmetabolism, lipid homeostasis, and/or lipid distribution, e.g.non-alcoholic liver disease, fatty liver, or various forms ofdyslipidemia, by targeting a gene involved in the regulation of lipidmetabolism, lipid homeostasis, and/or lipid distribution.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of aSCAP gene, as well as compositions and methods for treating diseases anddisorders caused by the expression of a SCAP gene, such as non-alcoholicliver disease, fatty liver, or various forms of dyslipidemia. Thepharmaceutical compositions of the invention comprise a dsRNA having anantisense strand comprising a region of complementarity which is lessthan 30 nucleotides in length, generally 19-24 nucleotides in length,and is substantially complementary to at least part of an RNA transcriptof a SCAP gene, together with a pharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of a SCAP gene, methods of using thepharmaceutical compositions to treat diseases caused by expression of aSCAP gene, vectors encoding dsRNAs of the invention, and cellscomprising such dsRNAs or vectors of the invention.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A”, “T” and “U” (irrespective of whether written in capitalor small letters) each generally stand for a nucleotide that containsguanine, cytosine, adenine, thymine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, thymine, and uracil may bereplaced by other moieties without substantially altering the basepairing properties of an oligonucleotide comprising a nucleotide bearingsuch replacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “SCAP” or “SCAP gene” refers to genes encoding SREBPactivating proteins, non-exhaustive examples of which are found underGenBank accession numbers NM 012235.2 (Homo sapiens), NM_(—)001001144.1(Mus musculus) and U67060 (Cricetus cricetus).

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a SCAP gene, including mRNA that is a product of RNA processing of aprimary transcription product. The target sequence of any given RNAiagent of the invention means an mRNA-sequence of X nucleotides that istargeted by the RNAi agent by virtue of the complementarity of theantisense strand of the RNAi agent to such sequence and to which theantisense strand may hybridize when brought into contact with the mRNA,wherein X is the number of nucleotides in the antisense strand plus thenumber of nucleotides in a single-stranded overhang of the sense strand,if any.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding a SCAP gene). For example, a polynucleotideis complementary to at least a part of a SCAP gene mRNA if the sequenceis substantially complementary to a non-interrupted portion of an mRNAencoding the SCAP gene.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where the two strands are part of onelarger molecule, and therefore are connected by an uninterrupted chainof nucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connecting RNAchain is referred to as a “hairpin loop”. Where the two strands areconnected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker”. The RNA strands may have thesame or a different number of nucleotides. The maximum number of basepairs is the number of nucleotides in the shortest strand of the dsRNAminus any overhangs that are present in the duplex. In addition to theduplex structure, a dsRNA may comprise one or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that has nonucleotide overhang at either end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus. Mostpreferably, the mismatches are located within 6, 5, 4, 3, or 2nucleotides of the 5′ terminus of the antisense strand and/or the 3′terminus of the sense strand.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to a SCAP gene, e.g. a human SCAP gene, herein refer to the atleast partial suppression of the expression of a SCAP gene, e.g. a humanSCAP gene, as manifested by a reduction of the amount of mRNAtranscribed from a SCAP gene which may be isolated from a first cell orgroup of cells in which a SCAP gene is transcribed and which has or havebeen treated such that the expression of a SCAP gene is inhibited, ascompared to a second cell or group of cells substantially identical tothe first cell or group of cells but which has or have not been sotreated (control cells). Preferably, the cells are primary hamsterhepatocytes. The degree of inhibition is usually expressed in terms of

${\frac{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} ) - ( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} )}{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} )} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to SCAP genetranscription, e.g. the amount of protein encoded by a SCAP gene whichis secreted by a cell, or found in solution after lysis of such cells,or the number of cells displaying a certain phenotype, e.g. surfaceexpression of LDL receptor, or lipid synthesis. In principle, SCAP genesilencing may be determined in any cell expressing the target, eitherconstitutively or by genomic engineering, and by any appropriate assay.However, when a reference is needed in order to determine whether agiven dsRNA inhibits the expression of a SCAP gene by a certain degreeand therefore is encompassed by the instant invention, the assaysprovided in the Examples below shall serve as such reference.

Generally, the expression of a SCAP gene shall be considered to be atleast partially suppressed, if the probability of the difference in theresults of measurements of SCAP mRNA content, or other functionalparameter, obtained from treated cells and control cells essentiallyresulting from random effects is less than 5%. In other words,expression of a SCAP gene shall be considered to be at least partiallysuppressed, if said difference is statistically significant.

For example, in certain instances, expression of a SCAP gene, e.g. ahuman SCAP gene, is suppressed by at least 20%, 25%, 35%, 40%, 45%, or50% by administration of the double-stranded oligonucleotide of theinvention. In some embodiment, a SCAP gene, e.g. a human SCAP gene, issuppressed by at least 55%, 60%, 65%, 70%, 75%, or 80% by administrationof the double-stranded oligonucleotide of the invention. In someembodiments, a SCAP gene, e.g. a human SCAP gene, is suppressed by atleast 85%, 90%, or 95% by administration of the double-strandedoligonucleotide of the invention. Table 5 provides values for inhibitionof SCAP expression using various dsRNA molecules of the invention.

As used herein in the context of SCAP expression, e.g. expression of ahuman SCAP, the terms “treat”, “treatment”, and the like, refer torelief from or alleviation of pathological processes mediated by SCAPexpression. In the context of the present invention insofar as itrelates to any of the other conditions recited herein below (other thanpathological processes mediated by SCAP expression), the terms “treat”,“treatment”, and the like mean to relieve or alleviate at least onesymptom associated with such condition, or to slow or reverse theprogression of such condition.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by SCAP expression or an overt symptomof pathological processes mediated by SCAP expression. The specificamount that is therapeutically effective can be readily determined byordinary medical practitioner, and may vary depending on factors knownin the art, such as, e.g. the type of pathological processes mediated bySCAP expression, the patient's history and age, the stage ofpathological processes mediated by SCAP expression, and theadministration of other anti-SCAP expression agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. Double-stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of a SCAP gene,e.g. a human SCAP gene, in a cell or mammal, wherein the dsRNA comprisesan antisense strand comprising a region of complementarity which iscomplementary to at least a part of an mRNA formed in the expression ofa SCAP gene, e.g. a human SCAP gene, and wherein the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length, and wherein said dsRNA, upon contact with a cellexpressing said SCAP gene, inhibits the expression of said SCAP gene byat least 20%. The dsRNA comprises two RNA strands that are sufficientlycomplementary to hybridize to form a duplex structure. One strand of thedsRNA (the antisense strand) comprises a region of complementarity thatis substantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of a SCAP gene, the other strand (the sense strand) comprisesa region which is complementary to the antisense strand, such that thetwo strands hybridize and form a duplex structure when combined undersuitable conditions. Generally, the duplex structure is between 15 and30, more generally between 18 and 25, yet more generally between 19 and24, and most generally between 19 and 21 base pairs in length.Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 19 and 24, and most generally between 19 and 21 nucleotides inlength. The dsRNA of the invention may further comprise one or moresingle-stranded nucleotide overhang(s).

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. In a preferred embodiment, a SCAP gene is the humanSCAP gene. In specific embodiments, the first strand of the dsRNAcomprises the sense strand sequences of the RNAi agents AD-9490-AD-9513(uneven numbers of the group of SEQ ID NO: 1-48, Table 1), and thesecond sequence is selected from the group consisting of the antisensestrand sequences of AD-9490-AD-9513 (even numbers of the group of SEQ IDNO: 1-48, Table 1).

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided above for theRNAi agents AD-9490-AD-9513 (Table 1). In other embodiments, the dsRNAcomprises at least two sequences selected from this group, wherein oneof the at least two sequences is complementary to another of the atleast two sequences, and one of the at least two sequences issubstantially complementary to a sequence of an mRNA generated in theexpression of a SCAP gene, e.g. a human SCAP gene. Generally, the dsRNAcomprises two oligonucleotides, wherein one oligonucleotide is describedas the sense strand in one of the RNAi agents AD-9490-AD-9513 (Table 1),and the second oligonucleotide is described as the antisense strand inone of the RNAi agents AD-9490-AD-9513 (Table 1).

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided for the RNAi agents AD-9490-AD-9513(Table 1), the dsRNAs of the invention can comprise at least one strandof a length of minimally 21 nt. It can be reasonably expected thatshorter dsRNAs comprising one of the sequences provided herein for theRNAi agents AD-9490-AD-9513 (Table 1), minus only a few nucleotides onone or both ends may be similarly effective as compared to the dsRNAsdescribed above. Hence, dsRNAs comprising a partial sequence of at least15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of thesequences of the RNAi agents AD-9490-AD-9513 (Table 1), and differing intheir ability to inhibit the expression of a SCAP gene, e.g. a humanSCAP gene, in an assay as described herein below by not more than 5, 10,15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence,are contemplated by the invention. Further dsRNAs that cleave within thetarget sequence of the RNAi agents AD-9490-AD-9513 (Table 1), canreadily be made using a SCAP mRNA sequence, e.g. a human SCAP mRNAsequence, e.g. GeneBank accession no. NM_(—)012235.2, and the respectivetarget sequence.

In addition, the RNAi agents provided in Table 1 identify a site in theSCAP mRNA that is susceptible to RNAi-effected cleavage. As such thepresent invention further includes RNAi agents that target within thesequence targeted by one of the agents of the present invention. As usedherein a second RNAi agent is said to target within the sequence of afirst RNAi agent if the second RNAi agent cleaves the message anywherewithin the mRNA that is complementary to the antisense strand of thefirst RNAi agent. Such a second agent will generally consist of at least15 contiguous nucleotides from one of the sequences provided in Table 1coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in a SCAP gene. For example, the3′-most 15 nucleotides of the target sequence of AD-9509 combined withthe next 6 nucleotides from the target SCAP gene produces a singlestrand agent of 21 nucleotides that is based on one of the sequencesprovided in Table 1.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity, and preferably from the 5′-end. For example, for a 23nucleotide dsRNA strand which is complementary to a region of a SCAPgene, the dsRNA generally does not contain any mismatch within thecentral 13 nucleotides. In another embodiment, the antisense strand ofthe dsRNA does not contain any mismatch in the region from positions 1,or 2, to positions 9, 10, 11, or 12, of the antisense strand (counting5′-3′). These positions are generally considered as the seed region(positions 1, or 2, to 9, or 10) and the site of mRNA cleavage(positions 11 and 12), respectively, and seem most sensitive tomismatches. The methods described within the invention can be used todetermine whether a dsRNA containing a mismatch to a target sequence iseffective in inhibiting the expression of a SCAP gene. Consideration ofthe efficacy of dsRNAs with mismatches in inhibiting expression of aSCAP gene is important, especially if the particular region ofcomplementarity in a SCAP gene is known to have polymorphic sequencevariation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Specific examples of preferred dsRNAcompounds useful in this invention include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Preferred modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an dsRNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular _CH_(2—)NH_CH₂—,—CH_(2—)N(CH₃)_O_CH_(2—)[known as a methylene (methylimino) or MMIbackbone], —CH_(2—)O_N(CH₃)_CH_(2—), —CH₂N(CH₃)_N(CH₃)_CH_(2—) and_N(CH₃)_CH_(2—)CH_(2—) [wherein the native phosphodiester backbone isrepresented as _O_P_O_CH_(2—)] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. Also preferred are dsRNAs having morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C_(I) to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O_CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O_CH_(2—)O_CH_(2—)N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T.and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. Cleavage of the RNAtarget can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the dsRNA of the invention and anysuitable promoter for expressing the dsRNA sequences. Suitable promotersinclude, for example, the U6 or H1 RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the dsRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver dsRNA of the invention to cells in vivo is discussed in moredetail below.

dsRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of a SCAP gene, such as pathological processesmediated by human SCAP expression, or diseases or disorders which can betreated by downregulation of SCAP expression. Such pharmaceuticalcompositions are formulated based on the mode of delivery. One exampleis compositions that are formulated for systemic administration viaparenteral delivery.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of a SCAP gene. The presentinventors have found that, because of their improved efficiency,compositions comprising the dsRNA of the invention can be administeredat surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogrambody weight of recipient per day is sufficient to inhibit or completelysuppress expression of a SCAP gene.

In general, a suitable dose of dsRNA will be in the range of 0.01microgram to 5.0 milligrams per kilogram body weight of the recipientper day, generally in the range of 1 microgram to 1 mg per kilogram bodyweight per day. The pharmaceutical composition may be administered oncedaily, or the dsRNA may be administered as two, three, or more sub-dosesat appropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful forvaginal delivery of agents, such as could be used with the agents of thepresent invention. In this embodiment, the dosage unit contains acorresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in genetics have generated a number of laboratory animal modelsfor the study of various human diseases, such as pathological processesmediated by SCAP expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical, pulmonary, e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal, oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the dsRNAs of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl ethanolamine=DOPE,dimyristoylphosphatidyl choline=DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol=DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl=DOTAP and dioleoylphosphatidylethanolamine=DOTMA). DsRNAs of the invention may be encapsulated withinliposomes or may form complexes thereto, in particular to cationicliposomes. Alternatively, dsRNAs may be complexed to lipids, inparticular to cationic lipids. Preferred fatty acids and esters includebut are not limited arachidonic acid, oleic acid, eicosanoic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which dsRNAs of the invention are administered inconjunction with one or more penetration enhancers, surfactants, andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsof the invention may be delivered orally, in granular form includingsprayed dried particles, or complexed to form micro or nanoparticles.DsRNA complexing agents include poly-amino acids; polyimines;polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S.application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions may be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions may containadditional components in addition to the dispersed phases, and theactive drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C₈-C₁₀glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids within the gastrointestinal tract, vagina,buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(m)1, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(m)1, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(m)1 or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B 1). Liposomes comprising a number of other lipid-polymerconjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212(both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomescomprising PEG-modified ceramide lipids are described in WO 96/10391(Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat.No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that canbe further derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include dsRNA. U.S. Pat.No. 5,665,710 to Rahman et al. describes certain methods ofencapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love etal. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make Transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁-C₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof; which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients:

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Pharmaceutical Compositions for the Delivery to the Respiratory Tract

Another aspect of the invention provides for the delivery of IRNA agentsto the respiratory tract. The respiratory tract includes the upperairways, including the oropharynx and larynx, followed by the lowerairways, which include the trachea followed by bifurcations into thebronchi and bronchioli. The upper and lower airways are called theconductive airways. The terminal bronchioli then divide into respiratorybronchioli which then lead to the ultimate respiratory zone, thealveoli, or deep lung. The deep lung, or alveoli, are the primary targetof inhaled therapeutic aerosols for systemic delivery of iRNA agents.

Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably the iRNAagent, within the dispersion can reach the lung where it can, forexample, be readily absorbed through the alveolar region directly intoblood circulation. Pulmonary delivery can be effective both for systemicdelivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations; administration by inhalation may be oral and/or nasal.Delivery can be achieved with liquid nebulizers, aerosol-based inhalers,and dry powder dispersion devices. Metered-dose devices are preferred.One of the benefits of using an atomizer or inhaler is that thepotential for contamination is minimized because the devices are selfcontained. Dry powder dispersion devices, for example, deliver drugsthat may be readily formulated as dry powders. An iRNA composition maybe stably stored as lyophilized or spray-dried powders by itself or incombination with suitable powder carriers. The delivery of a compositionfor inhalation can be mediated by a dosing timing element which caninclude a timer, a dose counter, time measuring device, or a timeindicator which when incorporated into the device enables dose tracking,compliance monitoring, and/or dose triggering to a patient duringadministration of the aerosol medicament.

Examples of pharmaceutical devices for aerosol delivery include metereddose inhalers (MDIs), dry powder inhalers (DPIs), and air-jetnebulizers. Exemplary delivery systems by inhalation which can bereadily adapted for delivery of the subject iRNA agents are describedin, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCTapplications WO98/31346; WO98/10796; WO00/27359; WO01/54664;WO02/060412. Other aerosol formulations that may be used for deliveringthe iRNA agents are described in U.S. Pat. Nos. 6,294,153; 6,344,194;6,071,497, and PCT applications WO02/066078; WO02/053190; WO01/60420;WO00/66206. Further, methods for delivering iRNA agents can be adaptedfrom those used in delivering other oligonucleotides (e.g., an antisenseoligonucleotide) by inhalation, such as described in Templin et al.,Antisense Nucleic Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al.,Expert Opin Biol Ther, 2001, 1:979-83; Sandrasagra et al., AntisenseNucleic Acid Drug Dev, 2002, 12:177-81.

The delivery of the inventive agents may also involve the administrationof so called “pro-drugs”, i.e. formulations or chemical modifications ofa therapeutic substance that require some form of processing ortransport by systems innate to the subject organism to release thetherapeutic substance, preferably at the site where its action isdesired; this latter embodiment may be used in conjunction with deliveryof the respiratory tract, but also together with other embodiments ofthe present invention. For example, the human lungs can remove orrapidly degrade hydrolytically cleavable deposited aerosols over periodsranging from minutes to hours. In the upper airways, ciliated epitheliacontribute to the “mucociliary excalator” by which particles are sweptfrom the airways toward the mouth. Pavia, D., “Lung MucociliaryClearance,” in Aerosols and the Lung: Clinical and Experimental Aspects,Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984. In thedeep lungs, alveolar macrophages are capable of phagocytosing particlessoon after their deposition. Warheit et al. Microscopy Res. Tech., 26:412-422 (1993); and Brain, J. D., “Physiology and Pathophysiology ofPulmonary Macrophages,” in The Reticuloendothelial System, S. M.Reichard and J. Filkins, Eds., Plenum, New. York., pp. 315-327, 1985.

In preferred embodiments, particularly where systemic dosing with theiRNA agent is desired, the aerosoled iRNA agents are formulated asmicroparticles. Microparticles having a diameter of between 0.5 and tenmicrons can penetrate the lungs, passing through most of the naturalbarriers. A diameter of less than ten microns is required to bypass thethroat; a diameter of 0.5 microns or greater is required to avoid beingexhaled.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more dsRNA agents and (b) one or more otherchemotherapeutic agents which function by a non-RNA interferencemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-dsRNAchemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions of the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs of the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by SCAP expression. In any event, theadministering physician can adjust the amount and timing of dsRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of a SCAP Gene

The invention relates in particular to the use of a dsRNA or apharmaceutical composition prepared therefrom for the treatment ofdisorders of lipid metabolism, lipid homeostasis, and/or lipiddistribution, e.g non-alcoholic liver disease, fatty liver,hyperlipemia, hyperlipidemia, hyperlipoproteinemia, hypercholesterolemiaand/or hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulindependent diabetes mellitus (NIDDM), coronary heart disease, obesity,metabolic syndrome, peripheral arterial disease, and cerebrovasculardisease. Owing to the inhibitory effect on SCAP expression, a dsRNAaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life of patients with such diseasesor disorders.

The invention furthermore relates to the use of an dsRNA or apharmaceutical composition thereof in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating diseases or disorders oflipid metabolism, lipid homeostasis, and/or lipid distribution, e.g.non-alcoholic liver disease, fatty liver, hyperlipemia, hyperlipidemia,hyperlipoproteinemia, hypercholesterolemia and/or hypertriglyceridemia,atherosclerosis, pancreatitis, non-insulin dependent diabetes mellitus(NIDDM), coronary heart disease, obesity, metabolic syndrome, peripheralarterial disease, and cerebrovascular disease. Where the pharmaceuticalcomposition aims for the treatment of diseases or disorders of lipidmetabolism, lipid homeostasis, and/or lipid distribution, preference isgiven to a combination with lipid lowering drugs, e.g. statins, insulinfor diabetes, and medication for liver disease.

Methods for Inhibiting Expression of a SCAP Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of a SCAP gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target SCAP gene, e.g. human SCAP, is silenced.Because of their high specificity, the dsRNAs of the inventionspecifically target RNAs (primary or processed) of the target SCAP gene.Compositions and methods for inhibiting the expression of these SCAPgenes using dsRNAs can be performed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of a SCAPgene, e.g. human SCAP, of the mammal to be treated. When the organism tobe treated is a mammal such as a human, the composition may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,subcutaneous, transdermal, airway (aerosol), nasal, rectal, vaginal andtopical (including buccal and sublingual) administration. In preferredembodiments, the compositions are administered by intravenous infusionor injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES

Design of siRNAs

siRNA design was carried out to identify siRNAs targeting hamster,mouse, and/or human SCAP. First, the mRNA sequences of Mus musculus(NM_(—)001001144.1) and Cricetus cricetus (U67060; Hua et al., Cell.1996, 87:415) SCAP were examined by computer analysis to identifyhomologous sequences of 19 nucleotides that yield RNAi agentscross-reactive between these two species.

In identifying agents cross-reactive between mouse and hamster, theselection was limited to 19mer sequences having at least 3 mismatches toany other sequence in the mouse genome by using a software tool providedby the Whitehead Institute at http://jura.wi.mit.edu/bioc/siRNAext/ inthe version of May 18, 2005. The sequences thus identified formed thebasis for the synthesis of the iRNA agents given in Table 2, whichcontain modified nucleotides. Therein, all pyrimidine-base bearingnucleotides in the sense strand, and all cytidines occurring in asequence context 5′-α-3′ and all uridines occurring in a sequencecontext 5′-ua-3′ in the antisense strand, are 2′-O-methyl-modifiednucleotides, and the 3′-terminal deoxythymidines

TABLE 2  RNAi agents selected for the down-regulation of mus musculus(NM_001001144.1) and Cricetus cricetus (U67060) SCAP,and minimal off-target interactions in mice SEQ SEQ Duplex ID IDidentifier Sense strand sequence¹ NO: Antisense strand sequence¹ NO:AL-DP-6054 ggcmgacmaumumacmcmumumgumacmaTT 49 ugumacmaaggumaaugucgccTT50 AL-DP-6055 gumcmcmumgumcmgaumcmgacmaumumcmTT 51gaaugucgaucgacmaggacTT 52 AL-DP-6056 cmacmumcmaaumggcmggumgagaumTT 53aucucmaccgccmauugagugTT 54 AL-DP-6057 umcmcmumgumcmgaumcmgacmaumumcmgTT55 cgaaugucgaucgacmaggaTT 56 AL-DP-6058 gagumgumcmumggcmumagcmgaumgTT 57cmaucgcumagccmagacmacucTT 58 AL-DP-6059cmumcmacmcmumgcmumumaaumcmgacmaTT 59 ugucgauumaagcmaggugagTT 60AL-DP-6060 ggaumumgumagcmumgcmumcmggcmumTT 61 agccgagcmagcumacmaauccTT62 AL-DP-6061 umumgumagcmumgcmumcmggcmumumaaTT 63uumaagccgagcmagcumacmaaTT 64 AL-DP-6062gcmumumaaumggumumcmcmcmumumgaumTT 65 aucmaagggaaccmauumaagcTT 66AL-DP-6063 acmacmumcmaaumggcmggumgagaTT 67 ucucmaccgccmauugaguguTT 68AL-DP-6064 acmcmumcmacmcmumgcmumumaaumcmgaTT 69 ucgauumaagcmaggugagguTT70 AL-DP-6065 gaggumgaagcmumumcmggaumumgTT 71 cmaauccgaagcuucmaccucTT 72¹Capital letters = desoxyribonucleotides; small letters =ribonucleotides; underlined: nucleoside thiophosphates; cm =2'-O-methyl-cytidine; um = 2'-O-methyl-uridine

In order to furthermore identify agents useful for inhibiting theexpression of human SCAP, the sequences of Homo sapiens(NM_(—)012235.2), Mus musculus (NM_(—)001001144.1) and Cricetus (U67060)SCAP were compared in a second step, and homologous sequences of 19nucleotides that yield RNAi agents cross-reactive between these threespecies were identified. To minimize non-specific effects in humans, theselection was further narrowed by fastA comparison to the gene sequencesin the human RefSeq database (available fromhttp://www.ncbi.nlm.nih.gov/RefSeq/), Rev. 17. The 24 19mer sequences ofthe RNAi agents in Table 1 were identified which had at least 2mismatches to any other gene in the human RefSeq database for both thesense and the antisense strand, wherein, for the closest matching gene,at least one of these mismatches came to lie in the “seed region” (Pos.2 to 10, counting 5′ to 3′), which is particularly sensitive tomismatches, and which had at least two mismatches, or at least one seedregion mismatch, to any other gene in the mouse RefSeq database.

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′, an appropriately modified solid support was used for RNAsynthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 2.

TABLE 3 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′-phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′-phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′-phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am2′-O-methyladenosine-5′-phosphate Cm 2′-O-methylcytidine-5′-phosphate Gm2′-O-methylguanosine-5′-phosphate Tm 2′-O-methyl-thymidine-5′-phosphateUm 2′-O-methyluridine-5′-phosphate Af2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, underlined:nucleoside-5′-phosphorothioate a, c, g, t, u am, cm, gm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate tm, um ^(a)capital lettersrepresent 2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

dsRNA Expression Vectors

In another aspect of the invention, Human SCAP specific dsRNA moleculesthat modulate Human SCAP gene expression activity are expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Thesetransgenes can be introduced as a linear construct, a circular plasmid,or a viral vector, which can be incorporated and inherited as atransgene integrated into the host genome. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single Human SCAP gene or multiple Human SCAP genes over a periodof a week or more are also contemplated by the invention. Successfulintroduction of the vectors of the invention into host cells can bemonitored using various known methods. For example, transienttransfection. can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection. ofex vivo cells can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

The Human SCAP specific dsRNA molecules can also be inserted intovectors and used as gene therapy vectors for human patients. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Effects of SCAP RNAi on the Genes Involved in Fatty Acid and CholesterolSynthesis in Primary Hepatocytes and in Livers from in vivo Experiments

Single Dose Screen in Primary Hamster Hepatocytes

Hepatocytes were isolated from a hamster liver and plated on 60-mmdishes at a density of 1.2×10⁶ cells/dish. After a 2-h attachmentperiod, cells were transfected with 200 nM of the indicated siRNA usingoligofectamine. Total RNA was isolated from the cells 24-h aftertransfection using RNA STAT-60 solution (Tel-Test Inc., Friendswood,Tex., USA). Ten μg of RNA each dish was treated with DNase I (DNA-free;Ambion Inc., Austin, Tex., USA). First-strand cDNA was synthesized from2 μg of DNase I-treated total RNA with random hexamer primers using theABI cDNA synthesis kit (N808-0234; PE Biosystems, Foster City, Calif.,USA). The following specific primers for each gene were designed usingPrimer Express software (PE Biosystems): β-actin, 5′ primer,5′-GGCTCCCAGCACCATGAA-3′, 3′ primer, 5′-GCCACCGATCCACACAGAGT-3′; SCAP,5′ primer, 5′-GTACCTGCAGATGATGTCCATTG-3′, 3′ primer,5′-CTGCCATCCCGGAAAGTG-3′ β-actin was used as the invariant control. Thereal-time RT-PCR reaction was set up in a final volume of 20 μlcontaining 20 ng of reverse-transcribed total RNA, 167 nM of the forwardand reverse primers, and 10 μl of 2×SYBR Green PCR Master Mix (4312704;PE Biosystems). PCR reactions were carried out in 384-well plates usingthe ABI PRISM 7900HT Sequence Detection System (PE Biosystems). Allreactions were done in triplicate. The relative amount of all mRNAs wascalculated using the comparative threshold cycle (C_(T)) method. Hamsterβ-actin mRNAs was used as the invariant controls. Values represent theamount of mRNA relative to that in untransfected cells, which is definedas 1 (n=1 plate).

TABLE 4 Screening siRNAs specific for inhibition of SCAP in mice andhamsters in hamster primary hepatocytes Duplex identifier remaining SCAPmRNA Mock transfection 1.0 (by definition) AL-DP-6054 1.06 AL-DP-60551.04 AL-DP-6056 1.59 AL-DP-6057 1.32 AL-DP-6058 1.06 AL-DP-6059 0.62AL-DP-6061 1.21 AL-DP-6062 0.22 AL-DP-6063 0.89 AL-DP-6064 0.81AL-DP-6065 0.31

TABLE 5 Screening human crossreactive siRNAs for inhibition of SCAP inhamster primary hepatocytes Duplex identifier remaining SCAP mRNA Mocktransfection 1.0 (by definition) AL-DP-6062 0.37 AD-9505 0.21 AD-94980.22 AD-9512 0.23 AD-9490 0.27 AD-9495 0.27 AD-9503 0.27 AD-9494 0.28AD-9500 0.28 AD-9492 0.31 AD-9499 0.33 AD-9496 0.34 AD-9510 0.37 AD-95110.38 AD-9491 0.41 AD-9506 0.42 AD-9508 0.44 AD-9502 0.45 AD-9504 0.51AD-9507 0.53 AD-9493 0.61 AD-9501 0.62 AD-9497 0.66 AD-9509 0.69 AD-95130.78Effects of SCAP RNAi on the Genes Involved in Fatty Acid and CholesterolSynthesis in Hamsters in vivo

For in vivo RNAi experiments, AL-DP-6062 formulated with liposomes wasinjected (4 mg/kg) into 6 hamsters via the jugular vein. Three daysafter injection, the animals were sacrificed, and total RNA was preparedfrom livers using established procedures.

Total RNA was prepared from the hepatocytes using an RNeasy kit fromQIAGEN (Valencia, Calif.). Ten μg of RNA from each hamster liver wastreated with DNase I (DNA-free; Ambion Inc., Austin, Tex., USA).First-strand cDNA was synthesized from 2 μg of DNase I-treated total RNAwith random hexamer primers using the ABI cDNA synthesis kit (N808-0234;PE Biosystems, Foster City, Calif., USA). Equal amounts of cDNA from 6hamsters were pooled. Specific primers for each gene were designed usingPrimer Express software (PE Biosystems) for the following genes:β-actin, 5′ primer, 5′-GGCTCCCAGCACCATGAA-3′, 3′ primer,5′-GCCACCGATCCACACAGAGT-3′; SCAP, 5′ primer,5′-GTACCTGCAGATGATGTCCATTG-3′, 3′ primer, 5′-CTGCCATCCCGGAAAGTG-3′;SREBP-1c, 5′ primer, 5′-ACGCAGTCTGGGCAACAA-3′, 3′ primer,5′-GAGCTGGAGCATGTCTTCAAAC-3′; SREBP-2, 5′ primer,5′-GTCAGCAATCAAGTGGGAGAGT-3′, 3′ primer, 5′-CTACCACCACCAGGGAAGGA-3′;Fatty acid synthase (FAS), 5′ primer, 5′-AACAAGCAATGCCAGCTCACT-3′, 3′primer, 5′-AACAGGCCCAAGCTTTGTTG-3′; stearoyl-CoA desaturase-1 (SCD-1),5′ primer, 5′-CAGAATGGACGGGAGAAGCA-3′, 3′ primer,5′-TCATTTCAGGGCGGATGTC-3′; HMG-CoA synthase, 5′ primer,5′-CCTATGACTGCATTGGGCG-3′, 3′ primer, 5′-CCCAGACTCCTCAAACAGCTG-3′;HMG-CoA reductase, 5′ primer, 5′-ACCATCTGTATGATGTCAATGAACA-3′, 3′primer, 5′-GCTCAATACGTCCTCTTCAAATTT-3′. The real-time RT-PCR reactionwas set up as described above. Hamster β-actin was used as the invariantcontrol. Values represent the amount of mRNA relative to the amount ofmRNA in livers of the hamsters injected with saline, which was definedas 1.

Protein Expression of SCAP and SREBPs in Livers of the Hamsters Injectedwith siRNA

Membrane and nuclear proteins were prepared from frozen liver asdescribed previously (Engelking et.al., J. Clin. Invest. 113: 1168-1175,2004). Equal amounts of protein were subjected to SDS-PAGE on 8% gelsand transferred to Hybond ECL membrane (Amersham). Immunoblot analyseswere performed using polyclonal anti-hamster SREBP-1 and SREBP-2antibodies (Shimomura et al., PNAS, 94: 12354-12359, 1997).Antibody-bound bands were detected using the SuperSignal CL-HRPsubstrate system (Pierce Biotechnology Inc., Rockford, Ill.). Anti-CREB(cAMP response element binding protein) and anti-RAP (receptorassociated protein) were used as loading controls for hepatic nuclearand membrane proteins, respectively. Signals were quantified using ImageJ program available from the Research Services Branch, NationalInstitute of Mental Health (Bethesda, Md.) and values represent theamount of protein relative to those in livers of the hamsters injectedwith saline which are defined as 1.

TABLE 6 mRNA expression of SCAP and other genes downstream of SCAP inhamsters 3 days after treatment with 4 mg/kg AL-DP-6062 (Hepatocytes: n= 2, Liver: pooled cDNA from 6 hamsters) Hepatocytes Liver SCAP 0.220.14 SREBP-1c 0.48 0.39 SREBP-2 0.65 0.51 FAS 0.78 0.68 SCD-1 0.74 0.86HMG-CoA synthase 0.34 0.47 HMG-CoA reductase 0.88 0.27

TABLE 7 mRNA and protein expression of SCAP in livers of the hamstersinjected with siRNA (pooled cDNA or pooled protein from 6 hamsters)siRNA SCAP Mrna SCAP protein Saline 1 1 Luciferase 1.07 0.9 SCAP 0.140.1 SCAP-MM 0.82 0.9

TABLE 8 mRNA and protein expression of SREBP-1 in livers of the hamstersinjected with siRNA (pooled cDNA or pooled protein from 6 hamsters) mRNAProtein siRNA SREBP-1c pSREBP-1 nSREBP-1 Saline 1 1 1 Luciferase 0.590.8 0.9 SCAP 0.38 0.4 0.4 SCAP-MM 0.68 0.9 0.8

TABLE 9 mRNA and protein expression of SREBP-2 in livers of the hamstersinjected with siRNA (pooled cDNA or pooled protein from 6 hamsters) mRNAProtein siRNA SREBP-2 pSREBP-2 nSREBP-2 Saline 1 1 1 Luciferase 0.95 10.7 SCAP 0.5 0.2 0.5 SCAP-MM 0.81 0.9 0.6

TABLE 10 Cholesterol and triglyceride concentrations in plasma andliver. (n = 6) Plasma (mg/dl) Liver (mg/g) RNAi cholesteroltriglycerides cholesterol triglycerides Saline 91 ± 5 166 ± 12 2.1 ± 0.13.6 ± 0.2 Luciferase 113 ± 8  130 ± 12 2.3 ± 0.1 3.9 ± 0.3 SCAP 105 ± 2 157 ± 18 2.3 ± 0.1 3.7 ± 0.1 SCAP-MM 119 ± 13 164 ± 25 2.6 ± 0.1 4.0 ±0.3

We claim:
 1. A double-stranded ribonucleic acid (dsRNA) for inhibitingthe expression of a human SCAP gene in a cell, wherein said dsRNAcomprises a sense strand comprising SEQ ID NO:1 and an antisense strandcomprising SEQ ID NO:2, and wherein said dsRNA is at least 15nucleotides in length and less than 30 nucleotides in length.
 2. ThedsRNA of claim 1, wherein said at least 20% inhibition of expression ofa SCAP gene is effected in primary hamster hepatocytes.
 3. The dsRNA ofclaim 1, wherein said dsRNA comprises at least one modified nucleotide.4. The dsRNA of claim 3, wherein said modified nucleotide is chosen fromthe group of: a 2′-O-methyl modified nucleotide, a nucleotide comprisinga 5′phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 5. ThedsRNA of claim 3, wherein said modified nucleotide is chosen from thegroup of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 6. A cellcomprising the dsRNA of claim
 1. 7. A pharmaceutical composition forinhibiting the expression of a human SCAP gene in an organism,comprising a dsRNA and a pharmaceutically acceptable carrier, whereinthe dsRNA comprises a sense strand comprising SEQ ID NO:1 and anantisense strand comprising SEQ ID NO:2, and wherein said dsRNA is atleast 15 nucleotides in length and less than 30 nucleotides in length.8. The pharmaceutical composition of claim 7, wherein said at least 20%inhibition of expression of a SCAP gene is effected in primary hamsterhepatocytes.
 9. A method for inhibiting the expression of a human SCAPgene in a cell, the method comprising: (a) introducing into the cell adouble-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises asense strand comprising SEQ ID NO:1 and an antisense strand comprisingSEQ ID NO:2, and wherein said dsRNA is at least 15 nucleotides in lengthand less than 30 nucleotides in length; and (b) maintaining the cellproduced in step (a) for a time sufficient to obtain degradation of amRNA transcript of a human SCAP gene, thereby inhibiting expression of ahuman SCAP gene in the cell.
 10. The method of claim 9, wherein thehuman SCAP gene is a Homo sapiens SCAP gene.
 11. A vector for inhibitingthe expression of a human SCAP gene in a cell, said vector comprising aregulatory sequence operably linked to a nucleotide sequence thatencodes at least one strand of a dsRNA, wherein one of the strands ofsaid dsRNA is substantially complementary to a strand chosen from thegroup consisting of a strand comprising SEQ ID NO:1 and a strandcomprising SEQ ID NO:2, and wherein said dsRNA is at least 15nucleotides in length and less than 30 nucleotides in length.
 12. A cellcomprising the vector of claim 11.